Many assay procedures utilize enzymes as catalysts for analytical reactions. In particular, the enzyme peroxidase is among the most commonly used in enzymatic detection reactions.
Peroxidases are widespread in nature, being found particularly in a variety of plants. They appear to catalyze the same reaction, but differ markedly in physicochemical and kinetic properties. Three main types of peroxidases have been identified: acidic peroxidases with very high carbohydrate content; neutral or slightly basic peroxidases of medium sugar content; and very basic peroxidases of low sugar content. Among these peroxidases, the C isozyme of horseradish peroxidase is most commonly encountered in biotechnology.
Horseradish peroxidase is popularly used in labeling haptens, antibodies, protein A/G, avidin/streptavidin and DNA for enzyme immunoassay, immunocytochemistry, immunoblot and DNA detection. Detection of enzyme activity in the assay procedures takes advantage of the reactive cooperation between the enzyme and a highly sensitive color-forming agent or chromatic compound. The chromatic compound is a hydrogen donor that changes color due to the loss of hydrogen atoms. The dehydrogenation occurs in the presence of an oxidizing agent, typically hydrogen peroxide (H.sub.2 O.sub.2). It is this oxidation reaction that is catalyzed by the peroxidase. Since a colored product results from the reaction catalyzed by the enzyme, the peroxidase activity is determined from the intensity of the color generated.
Thus, in a typical assay protocol, the peroxidase is first introduced to the test medium. Then the chromatic compound and H.sub.2 O.sub.2 are mixed to form a colorless substrate that is added to the medium to test for the enzyme. The chromatic compound as substrate undergoes an oxidation reaction when catalyzed by peroxidase in the presence of H.sub.2 O.sub.2. A colored product results from the removal of hydrogen from the chromatic compound. The colored product is easily measured by spectrophotometry.
Many different types of chromatic compounds have been used in peroxidase assay procedures. When choosing a chromatic compound to be used in combination with H.sub.2 O.sub.2 as a peroxidase substrate, it is desired that there be a high rate of oxidation and thus a high rate of color conversion. It is further desired that the product have a stable color for easy spectrophotometric measurement. Examples of known chromatic compounds suitable as hydrogen donors are o-dianisidine, guaiacol, 5-aminosalicylic acid, 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonate) (ABTS), o-phenylenediamine (OPD) and 1,2-benzenediamine. These chromatic compounds have certain disadvantages such as high basal oxidation or poor stability of oxidized product or are mutagenic. Accordingly, an alternative chromatic compound that overcomes these disadvantages is preferred.
One group of chromatic compounds found to overcome the disadvantages described above is of the benzidine type, particularly tetraalkybenzidine or salts thereof. The most successfully used compound of this type is 3,3',5,5'-tetramethylbenzidine (TMB). This compound was synthesized in 1974 and found to have sufficient color sensitivity for use in peroxidase assays and in addition has been shown to desirably be noncarcinogenic (Garner et al., Cancer Letters 1, 39-42 (1975); Garner et al., J. Forensic Sci. 21, 816-821 (1976)). These qualities have popularized TMB to become the chromatic compound of choice for use as a substrate for peroxidase assay (Bos et al , J. Immunoassay 2, 187-204 (1981)).
While TMB provides many desirable qualities for use as a chromatic compound, it has the disadvantage of low stability. Hydrogen peroxide has also long been known to be relatively unstable in solution. Accordingly, early use of the reagents as a combined substrate involved mixing them together at the time of the procedure and then introducing the combination to the test medium. Such a procedure is relatively inconvenient and has been known to lead to inadvertent mixing errors that directly result in incorrect test results.
Artisans have tried in the past to produce a combined chromatic compound/peroxide substrate in advance of the test procedure but the same low stability of the substrate mixture was found. More particularly, when the TMB and H.sub.2 O.sub.2 cannot coexist as a substrate formulation for extended periods of time. This is because of the spontaneous evolution of color occurring due to the decomposition of the peroxide to give oxygen radicals, that attract hydrogen atoms from the chromatic compound. This occurs even in the absence of a catalyst such as peroxidase. Thus the substrate solution becomes useless in a relatively short time period.
Attempts have been made to find a stabilizing agent that prevents the substrate solution containing both a chromatic compound and peroxide from deteriorating when prepared and stored in a single container. One such attempt is disclosed in U.S. Pat. No. 4,891,314 to Pauly et al. The stabilizing agent utilized in the Pauly formulation is penicillin. While the formulation including the penicillin showed improvement over the prior art by providing some stabilization of the substrate formulation, it is not without its drawbacks and limitations. More particularly, it is disclosed that the addition of penicillin provides only a relatively limited increase in stability for one to several weeks. It has also been found that color sensitivity can be improved over the formulation using penicillin as the stabilizing agent.
Thus a need is identified to find a formulation for use as a substrate for assaying peroxidase activity that contains a chromatic compound, peroxide and a stabilizing agent that provides increased stabilization and color sensitivity. The present invention is shown to satisfy that need.